The present invention relates to reagents and methods for drug screening, and more particularly, neuronal cells and tissue for screening potential Alzheimer therapeutics.
In 1907, Alois Alzheimer described the case of a 51-year-old woman with a rapidly degenerating memory who, after a swift deterioration, died severely demented four years later. This condition, which now bears Alzheimer""s name, describes a fatal degenerative dementing disorder with initial mild memory impairment that progresses unrelentingly to a total debilitating loss of mental and physical faculties. Following symptom onset, the course of the disease varies considerably from a few years to over 20 years, with a mean survival of approximately 8 years. M. A. Smith, xe2x80x9cAlzheimer Disease,xe2x80x9d Internat. Rev. Neurobiol. 42:1 (1998).
Alzheimer disease affects 10-15% of individuals over 65 years and up to 47% of individuals over the age of 80. In both clinical and autopsy series in the United States and Europe, Alzheimer disease accounts for approximately two-thirds of all dementias affecting elderly individuals. D. A. Evans et al., J. Am. Med. Assoc. 262: 2551 (1989).
The most common and distinctive lesions present with the diseased brain are the neuritic senile plaques and neurofibrillary tangles. The major protein component of senile plaque cores and vascular amyloid is a small polypeptide of approximately 4.2 kDa termed amyloid-xcex2. A significant fraction of this protein is found to be associated with the cytoskeleton, presumably through its interaction with the microtubule-associated xcfx84 (xe2x80x9ctauxe2x80x9d) protein. It is believed that the increased phosphorylated status of tau protein represents one of the earliest neuronal changes prior to the development of neurofibrillary tangles.
Unfortunately, because of the heterogeneity of the factors thought to be responsible for Alzheimer disease and the lack of an animal model displaying the full spectrum of pathological changes, successful pharmacological interventions have not been established. What is needed is an easy, reliable method to determine the safety and efficacy of candidate therapeutics for the treatment and/or prevention of Alzheimer disease.
Definitions
The term xe2x80x9cdrugxe2x80x9d as used herein, refers to any medicinal substance used in humans or other animals. Encompassed within this definition are compound analogs, naturally occurring, synthetic and recombinant pharmaceuticals, hormones, neurotransmitters, etc. The present invention contemplates screening test compounds to identify a useful drug for the treatment of Alzheimers.
Most current attempts at therapeutics for Alzheimer disease are directed at neurotransmitter deficiencies. The term xe2x80x9cneurotransmitterxe2x80x9d includes any compound which functions in the nervous system to result in the transmission of chemical signals between cells. Examples of neurotransmitters include, but are not limited to neuropeptides, acetocholine, and amino acids (e.g., GABA). Other compounds are also contemplated, including dopamine, norepinephrine, etc.
The term xe2x80x9cGABAxe2x80x9d refers to xcex3-aminobutyric acid, a major inhibitory neurotransmitter in both vertebrates and invertebrates. Kuffler and Edwards, J. Neurophysiol., 21:589 (1965; Otuska et al., Proc. Natl. Acad. Sci USA 56:1110 (1966); Usherwood and Grundfest, J. Neurophysiol., 28:497 (1965). The term xe2x80x9cGABA receptorsxe2x80x9d thus refers to structures expressed by cells and which recognize GABA.
The present invention contemplates the detection of a variety of therapeutic compounds, including but not limited to compounds that inhibit re-entry of neuronal cells into the cell cycle. Such compounds may be agonists or antagonists.
The term xe2x80x9cagonistxe2x80x9d refers to molecules or compounds which mimic the action of a xe2x80x9cnativexe2x80x9d or xe2x80x9cnaturalxe2x80x9d compound. Agonists may be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, agonists may be recognized by receptors expressed on cell surfaces. This recognition may result in physiologic and/or biochemical changes within the cell, such that the cell reacts to the presence of the agonist in the same manner as if the natural compound was present.
The term xe2x80x9cantagonistxe2x80x9d refers to molecules or compounds which inhibit the action of a xe2x80x9cnativexe2x80x9d or xe2x80x9cnaturalxe2x80x9d compound. Antagonists may or may not be homologous to these natural compounds in respect to conformation, charge or other characteristics. Thus, antagonists may be recognized by the same or different receptors that are recognized by an agonist. Antagonists may have allosteric effects which prevent the action of an agonist. Or, antagonists may prevent the function of the agonist.
The term xe2x80x9chost cellxe2x80x9d or xe2x80x9ccellxe2x80x9d refers to any cell which is used in any of the screening assays of the present invention. The present invention contemplates xe2x80x9chost cellsxe2x80x9d or xe2x80x9ccellsxe2x80x9d in their natural states as well as genetically altered cells.
As used in the present invention, the term xe2x80x9ctransformationxe2x80x9d refers to the introduction of foreign genetic material into a cell or organism. Transformation may be accomplished by any method known which permits the successful introduction of nucleic acids into cells and which results in the expression of the introduced nucleic acid. xe2x80x9cTransformationxe2x80x9d includes but is not limited to such methods as transfection, microinjection, electroporation, and lipofection (liposome-mediated gene transfer). Transformation may be accomplished through use of any expression vector.
The term xe2x80x9cgenexe2x80x9d refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor thereof. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity is retained. In the case of the present invention, oncogenes are contemplated and the desired activity is to cause neuronal cells and tissue to exhibit characteristics associated with re-entry into the cell cycle. Such characteristics may be cellular antigen expression (e.g. phospho-tau expression). On the other hand, a simple characteristic that can be readily measured is the incorporation of a nucleic acid precursor (indicating that the cells are traversing the S-phase of the cell cycle). Such precursors include, but are not limited to, 8-bromodeoxyuridine and tritiated-thymidine.
The term xe2x80x9cwild-typexe2x80x9d refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the xe2x80x9cnormalxe2x80x9d or xe2x80x9cwild-typexe2x80x9d form of the gene. In contrast, the term xe2x80x9cmodifiedxe2x80x9d or xe2x80x9cmutantxe2x80x9d refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. The present invention contemplates wild-type oncogenes (e.g. from tumors) as well as oncogenes generated by mutation.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, usually more than three (3), and typically more than ten (10) and up to one hundred (100) or more (although preferably between twenty and thirty). The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5xe2x80x2 phosphate of one mononucleotide pentose ring is attached to the 3xe2x80x2 oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the xe2x80x9c5xe2x80x2 endxe2x80x9d if its 5xe2x80x2 phosphate is not linked to the 3xe2x80x2 oxygen of a mononucleotide pentose ring and as the xe2x80x9c3xe2x80x2 endxe2x80x9d if its 3xe2x80x2 oxygen is not linked to a 5xe2x80x2 phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5xe2x80x2 and 3xe2x80x2 ends.
The present invention relates to reagents and methods for drug screening, and more particularly, neuronal cells and tissue for screening potential Alzheimer therapeutics. It is not intended that the present invention be limited by the nature of the cells employed. In one embodiment, the cells are primary neurons. It is preferred that the cell employed is mammalian.
The present invention contemplates the use of neuronal cells treated in a manner such that they re-enter the cell cycle. The present invention contemplates using these treated cells in compound screening assays. The neuronal cells may be treated using chemical agents added to culture media. In a preferred embodiment, the neuronal cells are treated by transfecting them with an expression vector comprising one or more oncogenes (e.g. a combination of oncogenes).
In one embodiment, the present invention contemplates a neuronal cell (or cell culture) that has been subjected to treatment so as to exhibit at least one characteristic associated with the re-entry into the cell cycle. It is not intended that the present invention be limited by the nature of the treatment. However, in one embodiment, said treatment comprises transfection with an expression vector comprising one or more oncogenes. It is also not intended that the present invention be limited by the particular characteristic exhibited. However, in one embodiment, said characteristic comprises increased phosphorylation of the tau protein.
In one embodiment, the present invention contemplates a compound screening method comprising: a) providing, in any order: i) a reaction vessel; ii) a plurality of neuronal cells treated such that they exhibit at least one characteristic associated with the re-entry into the cell cycle; iii) a test compound b) combining said cells and said compound within said reaction vessel under conditions such that said compound is free to interact with said cells; and c) detecting said interaction. Again, it is not intended that the present invention be limited by the nature of the treatment. However, in one embodiment, said treatment comprises transfection with an expression vector comprising one or more oncogenes. It is also not intended that the present invention be limited by the particular characteristic exhibited. However, in one embodiment, said characteristic comprises expression of the tau protein.
It is not intended that the present invention be limited by the nature of the reaction vessel. Such screening can be done in a microwell of a microtiter plate. Alternatively, newer, high-throughput screening formats can be used (e.g. silicon-based systems). In one embodiment, the present invention also contemplates determining the viability of the cells after contact with the test compound.